Physiological Process and Mechanism of Senescence in Mutant Rice with Functional Deletion on OsVHA-A1 Gene
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摘要: 负责编码水稻液泡膜ATP酶A亚基的OsVHA-A1基因在水稻生长发育和抵抗环境压力等诸多方面发挥重要作用。本试验以筛选纯合的OsVHA-A1基因缺失突变体株系及其野生型为材料，发现OsVHA-A1基因缺失突变会导致该突变体水稻生育后期发生严重的早衰现象与减产。两种基因型水稻叶片与根系中抗氧化保护酶活性随生育期呈现动态变化，孕穗期后突变体根系和叶片的衰老指标（包括H2O2含量、O2-产生率和MDA含量）均明显高于野生型；与野生型对比，突变体根系衰老程度显著高于其叶片。在早衰前后两个时期，即苗期和灌浆高峰期，对OsVHA-A1基因进行qRT-PCR定量，发现灌浆高峰期时的突变体与野生型叶片和根系中的OsVHA-A1基因相对于苗期均下调表达，V-ATPase酶活性也显著下降，突变体下降幅度大于野生型。利用16S扩增子分析根际土壤微生物组成时发现，灌浆高峰期突变体根际土壤中植物病原菌的含量显著上升。同时，HPLC技术测得突变体根际土壤中对羟基苯甲酸和对香豆酸两种酚酸类物质极显著低于野生型。说明不同基因型水稻根际土壤微生物存在对根系分泌物的选择性利用并诱导根际病原菌的增长。相关性分析结果表明，孕穗期后两种基因型水稻的根系活力下降变化与其功能叶片叶绿素含量及光合速率降低呈一定的相关关系。综上所述，OsVHA-A1基因缺失突变直接介导了水稻根系和叶片早衰的发生，并通过选择性诱导根际病原菌增长而加剧突变体根系与整体老化。Abstract: Vacuolar H+-ATPases (V-ATPase) is a multi-subunit enzyme complex found in the tonoplast of eukaryotes. V-ATPase subunitA(VHA) encoded by OsVHA-A1 gene is crucial in the development and tolerance to environmental stress of rice. This study applied a wild type and a purified mutant rice with a single cytosine deletion from its OsVHA-A1 gene to compare the differences of their phenotypes during the entire growth period. The mutant rice senesced at late-growth stage resulting a significant yield reduction as compared to the wild type. The activities of antioxidant enzymes including SOD, POD and CAT of the two isogene rice lines showed differential temporal patterns.The senescence-induced increases on O2- production as well as H2O2 and MDA contents in the leaves and roots of the mutant rice after booting stage were generally higher than those of the wild type.The performance of the roots tended to be poorer than that of the leaves in the mutant type in comparison with that of the wild type. OsVHA-A1 was significantly down-regulated and the V-ATPase activity significantly declined at grain-filling stage after senescence from seeding stage before senescence for both lines. But the down-regulated OsVHA-A1 expression and reduced V-ATPase activity in the roots and leaves were greater in the mutant rice than those in the wild type. It appeared that OsVHA-A1 played a crucial role in regulating rice senescence.Aside from genetic changes, environmental factors also contributed to the heightened root senescence, as the 16S rDNA sequencing showed that the pathogenic fungi population in the rhizosphere soil increased significantly when the mutant type at its grain-filling stage. In the soil, HPLC analysis identified 5 phenolic acids, i.e., phydroxybenzoic, vanillin, syringate, pcoumaric acid, and ferulic acid. The contents of phydroxybenzoic and pcoumaric acid were extremely significantly lower associated with the mutant than the wild type. It suggested that the pathogens might participate in the process of root senescence. Furthermore, the declined root activity was found correlating with the chlorophyll or photosynthesis decrease in the isogen rice lines after booting stage.It was concluded that the absence of functional OsVHA-A1 directly affected the senescence, whereas the altered underground microbial community further hastened the aging process of the rice plant.
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Key words:
- senescence /
- antioxidant enzymes /
- V-ATPase /
- rhizosphere microorganism /
- phenolic acid
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图 1 不同生育期OsVHA-A1基因缺失突变体与其野生型形态学特征
注：WT代表野生型，Mut代表突变体；A为分蘖期，B抽穗期。
Figure 1. Morphological phenotypes of OsVHA-A1 mutant and wild type rice at various development stages
图 3 OsVHA-A1突变体与野生型水稻叶片和根系中OsVHA-A1基因表达与V-ATPase酶活性
注：以苗期野生型叶片中OsVHA-A1基因表达量为对照组(CK)，采用2-△△Ct法计算基因相对表达量。
Figure 3. OsVHA-A1 expression and V-ATPase activity in leaves and roots of mutant and wild type rice
图 4 OsVHA-A1基因缺失突变体与其野生型根际土壤中的细菌和真菌(Groups)α多样性
注：A为细菌，B为真菌。
Figure 4. α-diversity indices based on observe species of fungi and bacteria in rhizosphere soil of wild and mutant type rice
图 5 OsVHA-A1基因缺失突变体与其野生型根际土壤中主要细菌与真菌群落结构(TOP 10)分析
注：A为细菌主要组成群落，B为真菌主要组成群落。
Figure 5. Bacterial and fungal communities in rhizosphere soil of wild and mutant type rice (P < 0.05)
图 6 不同浓度梯度酚酸标准品液相色谱
注：A为对羟基苯甲酸, B为香兰素，C为丁香酸, D为对香豆酸, E为阿魏酸。质量浓度梯度：6.250、3.125、1.563、0.781 mg·L-1。
Figure 6. HPLC standards for phenolic acid of varied concentrations
图 7 OsVHA-A1基因缺失突变体与其野生型水稻根际土壤酚酸液相色谱
注：1为对羟基苯甲酸t=11.57, 2为香兰素t=13.21，3为丁香酸t=14.78, 4为对香豆酸t=14.78, 5为阿魏酸t=30.57。
Figure 7. HPLC for phenolic acids in rhizosphere soil of mutant and wild type rice
图 8 灌浆高峰期OsVHA-A1基因缺失突变体与其野生型水稻根际土壤酚酸含量
Figure 8. Phenolic acid contents in rhizosphere soil of mutant and wild type rice
图 9 早衰后(孕穗期，抽穗期，灌浆高峰期)根系活力与叶绿素相关性分析
注：A为野生型，B为OsVHA-A1基因缺失突变体。图 10同。
Figure 9. Correlation between root activity and leaf chlorophyll content after senescence in different stages
图 10 早衰后(孕穗期，抽穗期，灌浆高峰期)根系活力与光合作用相关性分析
Figure 10. Correlation between root activity and leaf photosynthesis after senescence in different stages
表 1 OsVHA-A1基因缺失突变体与其野生型水稻农艺性状
Table 1. Agronomic traits of OsVHA-A1 mutant and wild type rice
水稻
类型株高
/cm根长
/cm地上部
/cm根干重
/g叶干重
/g有效穗
/穗结实率
/%千粒重
/g野生 127.3±2.5A 18.9±1.4a 108.5±2.1A 1.34±0.1A 4.75±1.5A 11.2±2.1A 88.3±4.6A 25.3±1.5A 突变 86.8±3B 15.8±1.2b 67.5±2.5B 0.73±0.1B 1.26±1.2B 5.8±1.5B 37.6±5.6B 13.5±1.4B 注：同列数据后不同大、小写字母分别表示差异达极显著(P＜0.01)和显著水平(P＜0.05)。 
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